Methods for Producing Antibodies

ABSTRACT

This invention provides methods for producing antibodies, wherein the methods comprise the step of administering an immunogen comprising both a target antigen and a background antigen to transgenic animals, into which a gene coding for the background antigen has been introduced. Since immunotolerance to the background antigens have thus been induced in the transgenic animals, the animals efficiently produce antibodies to target antigens.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.10/516,603, filed Jun. 8, 2005, which is a National Stage ofInternational Application No. PCT/JP03/07071, filed on Jun. 4, 2003, andclaims the benefit of priority of Japanese Application 2002-164834,filed Jun. 5, 2002 and Japanese Application 2002-180351, filed Jun. 20,2002. The contents of all of the foregoing applications are herebyincorporated by reference in their entireties.

TECHNICAL FIELD

This invention relates to methods for producing antibodies. Thisinvention also relates to the antibodies obtained by the methods of thisinvention. This invention further relates to transgenic non-humananimals useful for the production of antibodies generated by the methodsof this invention.

BACKGROUND ART

Antibodies are useful as therapeutic agents, diagnostic agents, orreagents for various diseases. Many kinds of antibodies have beenisolated to date. General methods for producing antibodies comprise thesteps of administering antigens to mammals such as mice; and obtainingantibodies derived from the serum of these animals. However, subjectantibodies are not always obtained efficiently by antibody productionmethods, as in the following cases, for example:

when a small quantity of antigen is used to immunize mammals; or

when an insufficiently purified antigen is used to immunize mammals.

Therefore, when immunizing, it is desirable to prepare a large quantityof a sufficiently purified antigen. Practically, however, many antigensare difficult to purify or to sufficiently prepare. Thus, the step ofantigen preparation has often prevented antibody production.

Membrane proteins are one example of antigens for which immunogens aredifficult to prepare. Generally, membrane proteins are often difficultto highly express or sufficiently purify. These difficulties have beenan obstacle in obtaining antibodies against membrane proteins.

Attention has been paid to methods that use baculoviruses to expresslarge quantities of membrane proteins. By introducing a gene thatencodes a subject membrane protein into a baculovirus genome, thesubject membrane protein is expressed on the membrane surface of thebudding baculovirus (WO 98/46777, Unexamined Published Japanese PatentApplication No. (JP-A) 2001-333773). Using these methods enablesexpression of a large quantity of a subject membrane protein on a viralmembrane surface.

However, in addition to exogenous membrane proteins, baculovirus-derivedmembrane proteins are also expressed on the membrane surface of thebaculoviruses thus obtained. Thus, when budding baculoviruses are usedas antigens, antibodies against baculovirus-derived membrane proteinsmay also be produced. Accordingly, it has been difficult to efficientlyproduce antibodies against subject membrane proteins by using knownimmunization methods.

For example, immunization using budding baculoviruses as antigens ofteninduces antibodies that recognize gp64. The membrane proteins ofbaculoviruses comprise large quantities of gp64. In addition, due togp64's high antigenicity, immunized animals can easily recognize gp64 as“nonself”. Consequently, budding baculoviruses can be thought topreferentially induce anti-gp64 antibodies.

Therefore, when using membrane proteins as antigens, the subjectmembrane proteins expressed on the baculovirus membrane surface must besufficiently purified. However, purifying exogenous membrane proteinsfrom budding baculoviruses is generally difficult. Thus, it can be saidthat sufficient quantities of highly purified membrane proteins cannotbe practically obtained for use in immunization. Using conventionalmethods to obtain target antibodies for these difficult-to-purifyantigens has been difficult.

DISCLOSURE OF THE INVENTION

An objective of the present invention is to solve the above-describedproblems. In other words, an objective of this invention is to providemethods for producing antibodies that enable target antibodies to beeasily obtained. In addition, an objective of this invention is toprovide transgenic non-human animals that efficiently produce subjectantibodies.

To solve the above-described problems, the inventors focused on antigensthat are comprised in immunogens and that interfere with the productionof subject antibodies. Then, the inventors thought that a subjectantibody could be easily obtained by using immunized animals whoseimmune response to such interfering antigens is repressed. Furthermore,the inventors found that the above-described problems can be solved byutilizing immunotolerance to the antigens that interfere with productionof the subject antibodies, to control the immune responses of theimmunized animals, and thereby complete the present invention.

Specifically, the present invention relates to methods for producingantibodies, transgenic non-human animals useful for these methods, andmethods for producing these non-human animals. Specifically, the presentinvention provides the following:

[1] A method for producing an antibody that recognizes a target antigen,wherein the method comprises the steps of:

i) immunizing a non-human animal that has immunotolerance to abackground antigen comprised in an immunogen, wherein the immunogencomprises both the target antigen and the background antigen; andii) obtaining an antibody against the target antigen, or a gene encodingthe antibody.

[2] The method of [1], wherein immunotolerance is induced artificially.

[3] The method of [1], wherein the non-human animal is a transgenicnon-human animal.

[4] A method for producing an antibody against a target antigen, whereinthe method comprises the steps of:

(a) preparing an immunogen comprising the target antigen and abackground antigen;(b) producing a transgenic non-human animal comprising a geneexpressibly encoding the background antigen;(c) administering the immunogen of (a) to the transgenic non-humananimal of (b); and(d) isolating the antibody against the target antigen from thetransgenic non-human animal.

[5] The method of [4], wherein the immunogen is a virus particle or apart thereof.

[6] The method of [5], wherein the virus is a baculovirus.

[7] The method of [4], wherein the target antigen is a membrane protein.

[8] The method of [6], wherein the background antigen is gp64.

[9] The method of [4], wherein the non-human animal is a mouse.

[10] An antibody that is produced by the method of any one of [1] to[9].

[11] A chimeric antibody between a non-human animal and human, or ahumanized antibody, produced using the antibody of [10].

[12] A transgenic non-human animal, into which a gene encoding a viralenvelope protein is introduced.

[13] The transgenic non-human animal of [12], wherein the virus is abaculovirus.

[14] The non-human animal of [13], wherein the viral envelope protein isgp64.

[15] The non-human animal of [12], wherein the non-human animal is amouse.

[16] The non-human animal of [12] for use in producing an antibodyagainst an antigen comprising a viral protein.

[17] A method for producing a non-human immunized animal, wherein themethod comprises the step of producing a transgenic non-human animalinto which a gene encoding a background antigen is introduced.

[18] A non-human immunized animal for obtaining an antibody against atarget antigen comprising a background antigen, wherein the animal isproduced by the method of [17].

[19] A method for producing an antibody against PepT1, wherein themethod comprises the steps of:

(a) preparing a baculovirus that expressibly comprises a DNA whichencodes PepT1 or a fragment thereof;(b) infecting a host cell with the baculovirus of (a) to obtain abudding virus that expresses PepT1 or a fragment thereof;(c) producing a transgenic non-human animal that expressibly comprises agene encoding a baculovirus membrane protein gp64;(d) immunizing the transgenic non-human animal of (c) with a fractioncomprising the budding virus of (b) or PepT1 or its fragment; and(e) recovering the antibody-recognizing PepT1 from the immunized animal.

This invention relates to methods for producing antibodies whichrecognize target antigens, wherein the methods comprise the step ofimmunizing immunogens that comprise a target antigen and a backgroundantigen, to non-human animals with immunotolerance to the backgroundantigen comprised in the immunogen.

The term “target antigen” denotes antigens recognized by subjectantibodies. The target antigens can be selected from any compoundcomprising antigenicity. Specifically, proteins, sugar chains, lipids,or inorganic substances are known to comprise antigenicity. The targetantigens may be naturally occurring or artificially synthesized. Theartificially synthesized target antigens comprise recombinant proteinsprepared by genetic engineering technology, and many kinds ofchemically-synthesized organic compounds.

According to the present invention, the term “background antigen”denotes substances comprising antigenic determinants for which antibodygeneration is not desired, or denotes the antigenic determinantsthemselves. For example, any antigenic substance that is not a targetantigen, but which is contaminated within the target antigen, is abackground antigen. Typical background antigens are proteinscontaminated within crudely purified target antigens. More specifically,host cell-derived proteins in a recombinant protein are examples ofbackground antigens. The term “background antigen” may also be definedto mean antigens that are comprised within an immunogen for inducingsubject antibody generation, and that induce production of a non-subjectantibody.

Generally, a background antigen is an antigenic substance other than atarget antigen. According to the present invention, however, antigenicdeterminants present on target antigen molecules may also be referred toas background antigens. For example, if an antigenic determinant forwhich antibody generation is undesired is present on a target antigenmolecule, the antigenic determinant is defined as a background antigen.Moreover, the background antigens of the present invention includesubstances that comprise antigenic determinants as background antigens,yet do not comprise target antigens.

According to the present invention, preferable background antigens areproteins, peptides, sugars, or glycoproteins. Of these, proteins orpeptides are particularly preferable background antigens. The term“peptide” denotes, for example, polypeptides that consist of 100 orfewer amino acid residues. The term “protein” includes “peptide”.

According to the present invention, the term “immunotolerance” denotes acondition in which an immune response, specific to an antigen that is animmunotolerance target (an immunotolerance antigen), is lost ordecreased. When the level of a subject's immune response to animmunotolerance antigen is reduced compared to that of a normalimmunized animal, the subject can be regarded to compriseimmunotolerance against the immunotolerance antigen. For example, whenthe amount of an antibody generated against an immunotolerance antigenis decreased in response to the administration of an immunotoleranceantigen, the level of immune response is then considered to be low.Immunotolerance levels are not limited.

In addition, the term “immunotolerance antigen” denotes antigenicsubstances for which a subject's immune response is decreased. Also,according to the present invention, a reduced level of a specific immuneresponse to an immunotolerance antigen denotes that the degree by whichthe immune response to the immunotolerance antigen has decreased isgreater than for other antigens. Thus, even if the immune response toantigens other than the immunotolerance antigen is decreased,immunotolerance has been established if the level of decrease in immuneresponse to other antigens is less than the level of decrease for theimmunotolerance antigen. Also, according to the present invention,immunotolerance includes cases of immunotolerance to antigenicsubstances other than background antigens. Subjects which areimmunotolerant to multiple antigens may also be used in the presentinvention, as long as they have an immune response to a target antigen.On the other hand, cases of immunodeficiency where the level of actualimmune response is decreased are not preferable, since generation ofantibodies against target antigens cannot be expected.

In the present invention, non-human animals that compriseimmunotolerance to a background antigen are used as immunized animals.Non-human animals comprising artificially induced immunotolerance arepreferably used. For example, non-human animals comprisingimmunotolerance can be generated as below:

First, a gene encoding a background antigen can be introduced into anon-human animal to generate a transgenic animal that comprises the geneencoding the background antigen. Transgenic animals thus obtained haveimmunotolerance to the expression product of the introduced gene (thebackground antigen). Immunotolerance can also be induced by multipleadministrations of an immunotolerance antigen (a background antigen) tonon-human animals in the fetal stage or shortly after birth.

Methods for administering antigens as immunotolerance antigens tonon-human animals in the fetal stage or shortly after birth can includemethods for administering the antigenic substances themselves intonon-human animals. Alternatively, indirect methods for administeringimmunotolerance antigens may also be applied. For example, subjectimmunotolerance antigens are administered by in vivo expression of genesthat encode the immunotolerance antigens. Such methods include methodsfor directly administering an antigen-coding DNA (naked DNA methods),for transplanting antigen-expressing cells into non-human animals,methods using viral vectors, and methods using DNA vaccines.

Of these methods, transgenic non-human animals which preserve a geneencoding an immunotolerance antigen in an expressible state arepreferred as the non-human animals comprising immunotolerance of thepresent invention. The transgenic animals comprise in their body animmunotolerance antigen that was originally an exogenous protein priorto the maturation of immune functions. Therefore, it is highly possiblethat the immune functions of the transgenic animals recognize theimmunotolerance antigen as being completely endogenous. Thus, the use ofsuch transgenic non-human animals is advantageous in inducingimmunotolerance in the present invention. The transgenic animals, intowhich immunotolerance antigens are introduced, produce few antibodies toimmunotolerance antigens, as shown in Example 8.

In addition, the immunotolerant traits of the transgenic animals can beinherited by their progeny. Therefore, once a transgenic non-humananimal has been established for the present invention, immunized animalscomprising the same traits can be stably provided.

This invention also relates to transgenic non-human animals into whichgenes encoding a viral envelope protein are introduced to produceantibodies against antigens comprising the viral proteins. Moreover,this invention relates to use of transgenic non-human animals in which agene encoding the viral envelope protein is expressibly maintained, asimmunized animals for producing antibodies against antigens comprisingviral envelope proteins. Furthermore, this invention relates to methodsfor producing non-human immunized animals, where the methods comprisethe step of generating a transgenic non-human animal into which a geneencoding a background antigen has been introduced.

Many kinds of transgenic non-human animals into which different kinds ofgenes have been introduced are known in the art. However, animals intowhich an exogenous gene encoding a background antigen has beenintroduced are not known to be useful as immune animals for a targetantigen that comprises a background antigen.

Since an animal in which a gene that encodes a target antigen proteinhas been deleted, (so called knock-out animals), does not comprise thetarget antigen protein congenitally, an antibody against the targetantigen can be obtained by administering the target antigen to theknock-out animal, even if the target antigen is highly homologous to aprotein present in the immunized animal. Moreover, it is possible toobtain an animal which is deficient in a target antigen, and whichexpresses an immunotolerant antigen, by crossing the targetantigen-deficient animal with the transgenic animal of the presentinvention.

Genes coding for background antigens can also be introduced into a fetalor post-fetal non-human animal in a fetal period or thereafter, by usingnaked DNA methods, DNA vaccine methods, or methods for transplantingcells that express background antigens. Non-human animals thus obtainedare also included in the transgenic non-human animals of the presentinvention.

There is no limitation as to the number of background antigens used toinduce immunotolerance in the immunotolerant non-human animals of thepresent invention. That is, a non-human animal, in which immunotoleranceto at least one background antigen has been induced, can be used in anantibody-production method of the present invention. Non-human animalsin which immunotolerance to multiple background antigens has beeninduced can also be used as immunized animals.

In the immunized animals, it is not always important to suppressproduction of antibodies against all of the background antigens thatmight be comprised in an immunogen. Production of antibodies thatrecognize background antigens is acceptable as long as they do notinterfere with the production and isolation of an antibody against atarget antigen. Therefore, for example, an immunized animal in whichimmunotolerance has only been induced to a major background antigen canbe used as a preferable immunized animal in the present invention.

In the present invention, non-human animals comprise, for example,monkeys, pigs, dogs, rats, mice, and rabbits. For example, rodents suchas rats, mice, and hamsters are preferable as non-human animals. Toinduce immunotolerance by preparing transgenic animals, it isadvantageous to use non-human animals which mature fast and for whichgene manipulation technologies have been established, such as rodents.Mice in particular are non-human animals that meet these requirements ata high level.

This invention relates to transgenic non-human animals into which genescoding for viral envelope proteins have been introduced. Transgenicnon-human animals of the present invention are useful in immunizationagainst a target antigen in the presence of viral envelope proteins.Typically, viral envelope proteins in this invention are proteins thatmake up an envelope of a budding virus. In baculoviruses, for example,the protein called gp64 is an envelope protein.

For example, a transgenic non-human animal with immunotolerance to thebaculoviral gp64 is useful as an immune animal for an immunogen producedby the baculovirus expression system. Many kinds of proteins can beproduced by the baculovirus expression system. Therefore, by using thesetransgenic animals and baculovirus expression systems in combination,target antibodies can be easily obtained by using a variety of proteinantigens as target antigens.

Immunogens of the present invention comprise both target antigens andbackground antigens. As described above, there is no particularlimitation as to the substances constituting target antigens orbackground antigens. When an animal with immunotolerance is produced byintroduction of a gene encoding a background antigen, the backgroundantigen is a protein. Immunogens may include substances other thantarget antigens and background antigens.

Furthermore, there is no limitation as to the types of backgroundantigens that comprise the immunogens of the present invention.Therefore, immunogens comprising multiple kinds of background antigens,which may interfere with the production of antibodies against a targetantigen, can also be used in the present invention. The presence ofthese background antigens is not a problem, as long as an immunizedanimal shows immunotolerance to each background antigen. Alternatively,background antigens which do not substantially interfere with theproduction of antibodies against a target antigen may be comprised in animmunogen, regardless of whether or not an immunized animal isimmunotolerant to them.

Generally, a target antigen comprises substances derived from biologicalmaterials. Biological materials are complex mixtures comprising variouscomponents. Thus, target antigens are usually prepared using variousmixtures as starting materials. Therefore, it is difficult to obtainhighly-purified target antigens. In other words, it involves a lot oftime and effort to isolate a large quantity of a highly pure targetantigen. Practically, it is almost inevitable that an immunogen containssubstances other than the target antigen.

Immunogens of the present invention specifically include cells, cellcultures, cell lysates, viruses, or unpurified antigens. Parts of cellsor viruses can be used as immunogens, as well as whole cells or wholeviruses. For example, cell membranes or virus envelopes can be used asimmunogens. When a cell or virus is used as an immunogen, a gene codingfor a subject antigen can be artificially introduced into the cell orvirus by recombinant gene technology that artificially expresses thesubject antigen.

One preferable immunogen of the present invention is a viral particle orpart thereof. Viruses are comprised of relatively simple components,including nucleic acids, and limited proteins, saccharides, and such.Consequently, the types of background antigens that may interfere withtarget antigen isolation are also limited. In sum, inducingimmunotolerance against a limited number of background antigens in ananimal to be immunized would be enough to carry out a method forproducing antigen of the present invention.

In the present invention, baculoviruses, for example, are preferredamong the viruses that can be used as immunogens. Baculoviruses areinsect viruses that comprise a structure whereby a double-stranded DNAgenome is covered with a capsid protein. Expression systems usingNucleopolyhedrovirus (NPV), a type of baculovirus, are useful as systemsfor expressing exogenous genes. NPV comprises strong promoter activity.Therefore, any protein can be produced in large quantities by insertingan exogenous gene into the NPV genome. Specifically, strong expressionof any exogenous gene is induced by recombinantly substituting the genecoding for the protein called polyhedron with the exogenous gene.

Any exogenous genes can be introduced into a baculovirus. For example, agene encoding a membrane protein can be used as an exogenous gene.

By using baculoviruses, a subject membrane protein can be expressedalong with a viral envelope protein in a form that retains thatstructure. Another big advantage of the baculovirus expression system isthat the expressed products are easily recovered as budding viralparticles.

Membrane proteins include many biologically important molecules, such asreceptors and transporters. However, many membrane proteins maintaintheir structure by being located in a cell membrane. In addition,membrane proteins are often post-translationally modified with sugarchains or lipids. Therefore, there are often cases where expressionsystems utilizing prokaryotes such as E. coli cannot reproduce membraneproteins in their in situ structure.

As methods for expressing exogenous proteins such as membrane proteinson viral envelopes, for example, the method of WO98/46777 or Loisel etal., for expressing envelope proteins using budding baculoviruses can beused (Loisel, T. P. et al., Nature Biotech. 15: 1300-1304 (1997)). Morespecifically, a recombinant vector for insect cells comprising a geneencoding an exogenous protein is constructed, and inserted, along withbaculoviral DNA, into insect cells such as Sf9. The exogenous proteinencoded by the recombinant vector is then expressed on mature viralparticles (virions), which are released by infected cells to the outsideof cells prior to infected cell death. Recombinant viruses that expressthe exogenous protein can thus be obtained.

In the present invention, a budding virus is a virus that is releasedfrom infected cells by budding. Generally, viruses covered with anenvelope can bud from cells infected with these viruses, and arereleased continuously, even when the cells have not been destroyed. Onthe other hand, adenoviruses that are not covered by an envelope, andherpes viruses that are covered by a nuclear envelope, are released fromthe cells all at once, upon cell destruction. Budding viruses areparticularly preferable in the present invention. In addition, thoseskilled in the art can suitably select hosts to be infected with arecombinant virus, depending on the type of virus used, so long as viralreplication is possible in the host. For example, insect Sf9 cells canbe used when using baculoviruses. Generally, protein expression systemsusing baculoviruses and insect cells can be useful because modificationssuch as fatty acid acetylation or glycosylation are carried out at thesame time as translation or post-translation, in the same way as inmammalian cells. In addition, the expression level of heterologousproteins in such systems is greater than that in mammalian cell systems(Luckow V. A. and Summers M. D., Virol. 167: 56 (1988)).

The viruses expressing exogenous proteins can be obtained by, forexample, culturing a host that has been infected with a recombinantvirus comprising a gene that encodes an exogenous protein.Alternatively, using methods such as the above-mentioned methods of W098/46777 and Loisel et al (Loisel, T. P. et al., Nature Biotech. 15:1300-1304 (1997)), a recombinant vector encoding an exogenous proteincan be inserted into an insect cell along with a baculovirus, andexogenous proteins can be expressed on the envelope of the baculovirusreleased outside of the cell. In addition, using methods like that ofStrehlow et al. (D. Strehlow et al., Proc. Natl. Acad. Sci. USA. 97:4209-4214 (2000)), packaging cells such as PA317 can be infected withrecombinant Moloney murine leukemia viruses, which are constructed usingvectors derived from Moloney viruses introduced with exogenousprotein-encoding genes, and the exogenous proteins can be expressed onthe envelope of viruses released outside of the cells. However, theviruses of the present invention that express exogenous proteins, usefulas immunogens, are not limited to those that are constructed using theabove methods.

Recombinant viruses constructed as described above can be purified usingknown methods. For example, known methods for purifying viruses includeaugmented density gradient centrifugation (Albrechtsen et al, J.Virological Methods 28: 245-256 (1990); Hewish et al., J. VirologicalMethods 7: 223-228 (1983)), size exclusion chromatography (Hjorth andMereno-Lopez, J. Virological Methods 5: 151-158 (1982); Crooks et al.,J. Chrom. 502: 59-68 (1990); Mento S. J. (Viagene, Inc.) 1994Williamsburg Bioprocessing Conference), affinity chromatography usingmonoclonal antibodies, sulphated fucose-containing polysaccharides andthe like (Najayou at al., J. Virological Methods 32: 67-77 (1991); Diacoet al., J. Gen. Virol. 67: 345-351 (1986); Fowler, J. VirologicalMethods 11: 59-74 (1986); TOKUSAIHYOU No. 97/032010 (UnexaminedPublication of Japanese National Phase Patent Application)), and DEAEion exchange chromatography (Haruna et al., Virology 13: 264-267(1961)). Thus, purification can be carried out using the above methodsor combinations thereof.

In the present invention, there is no limitation as to the kind ofbackground antigen which becomes an immunotolerance antigen for use asan antigen to induce immunotolerance in immune animals. Preferably, theimmunotolerance antigens are such substances that are comprised in animmunogen in a large quantity, or that have a strong antigenicity. Forexample, when a baculovirus is used as an immunogen, gp64 is preferablyused as an immunotolerance antigen. Gp64 is a major background antigen,which is expressed in large quantities on the surface of the viralenvelope, and which is susceptible to being recognized as non-self byanimals immunized with baculoviruses.

Baculoviruses comprise characteristics that are preferable in anexpression system for exogenous proteins. On the other hand, use of anexpression product produced by this system as an immunogen, it isaccompanied by production of background antigens, which can be adrawback. In particular, when using a baculovirus expression system toproduce a membrane protein that is used as an immunogen, the presence ofgp64 is a big problem. gp64 is comprised in large amounts in viralenvelope proteins. Thus, contamination of an exogenous membrane proteinwith gp64 is inevitable.

By using the antibody-production methods of the present invention, theinhibitory effect that background antigens have on the acquisition ofantibodies against a target antigen can be suppressed. Consequently, theuse of this invention enables sufficient application of the advantagesof a baculovirus expression system as an exogenous protein expressionsystem, even in the preparation of immunogens.

In the present invention, naturally occurring viruses or parts thereofcan also be used as immunogens. Development of an antibody thatrecognizes a specific antigenic determinant of a naturally occurringvirus is important to the specific detection of the virus, and also toprevention of or therapy for infection by that virus. Whereas antibodiesagainst major antigens can be easily produced, it is often difficult toacquire an antibody that recognizes a specific antigenic determinant.This situation is common to the above described case in which abaculovirus expression product is used as an immunogen.

When using a naturally occurring virus as an immunogen of the presentinvention, a gene coding for a protein that will act as a backgroundantigen, selected from proteins that constitute the virus, is introducedinto a non-human animal to prepare a transgenic animal. Alternatively,viral particles themselves, or parts thereof that comprise a targetantigen, are used as immunogens. In this way, an antibody thatrecognizes a target antigen can be efficiently obtained.

For example, the surface antigens of influenza viruses are importantantigens that determine the viral strain. If antibodies that recognizedthe surface antigens specific to each influenza virus strain could beeasily obtained, this would be useful to identification of the virus, aswell as in the prevention of or therapy for infection by the virus.However, when using the viral particles themselves as immunogens,antibodies that recognize structures common to the viruses will also beproduced in large quantities.

Antibodies that recognize surface antigens specific to each viral straincan be efficiently obtained by using a transgenic non-human animal ofthe present invention, which has immunotolerance to an envelope proteinthat is common to the influenza viruses. In other words, this inventioncan be also carried out using surface antigens that are specific to eachstrain of a virus as target antigens, and using structures that arecommon to the viruses as background antigens.

A preferable embodiment of the antibody-production methods of thepresent invention is described below. In this embodiment, membraneproteins are used as target antigens. For example, human-derivedmembrane proteins can be used as the membrane proteins.

First of all, a target protein is expressed on the surface of thebaculovirus envelope, and this baculovirus is used as an immunogen. As amethod for expression the membrane protein using baculoviruses, forexample, the methods for expressing membrane proteins using buddingbaculoviruses, disclosed in WO 98/46777, JP-A 2001-333773, Loisel et al.(T. P. Loisel et al., Nature Biotech. 15: 1300-1304 (1997)), can beused.

In more detail, a recombinant vector for insect cells is constructed tocomprise a gene encoding a membrane protein. This vector is thenintroduced into insect cells along with the baculovirus DNA. Sf9 cellsand such are used as the insect cells. The membrane protein encoded bythe recombinant vector is expressed in mature viral particles (virions)released extracellularly from the infected cells prior to cell death.Therefore, budding baculovirus particles that express the membraneprotein (target antigen) may be obtained by harvesting mature virusparticles. Methods for recovering budding baculovirus from culturedcells are also known in the art. The thus obtained budding baculovirusesthat express a membrane protein (target antigen) are used as immunogensof the present invention.

As described above, the surface of the baculovirus envelope expressesnot only a membrane protein (the target antigen), but also anotherenvelope protein derived from a baculovirus. In particular, gp64 isexpressed in large quantities on the surface of baculoviruses, and alsohas strong antigenicity. Therefore, when immunization is carried outusing a budding baculovirus, anti-gp64 antibodies are also produced, andthus antibodies to the membrane protein (target antigen) cannot beefficiently obtained.

Accordingly, in the present invention, an animal that expresses gp64 isused as an animal to be immunized. Specifically, a transgenic animalthat expresses gp64 is produced by introducing a vector that comprises agene encoding gp64 into an animal. The transgenic animals of the presentinvention are non-human animals. For example, a transgenic mouse intowhich the gp64 gene has been introduced can be used as an animal to beimmunized in the present invention.

In the present invention, these transgenic mice are immunized with thebudding baculovirus particles obtained as described above. Since thegp64-expressing transgenic mice endogenously express gp64, they compriseimmunotolerance to gp64, which acts as a background antigen. In otherwords, production of anti-gp64 antibodies in the gp64-expressingtransgenic mice is suppressed when the mice are immunized with thebudding baculovirus particles. As a result, antibodies against a targetmembrane protein can be produced efficiently.

Methods for producing transgenic mice are known in the art. For example,transgenic mice can be obtained according to the methods described inProc. Natl. Acad. Sci. USA 77: 7380-7384 (1980). Specifically, subjectgenes are introduced into mammalian totipotent cells, and then the cellsare brought up into individuals. A subject transgenic mouse can beobtained from the individuals thus obtained by screening for individualsin which the introduced gene has been integrated into both somatic cellsand germ cells. Fertilized eggs, early embryos, and cultured cells withmultipotency such as ES cells, and such, can be used as the totipotentcells for introducing a gene.

More specifically, transgenic mice can be prepared, for example, by themethod in Example 2.

The antibody-production methods of the present invention can be used toproduce polyclonal and monoclonal antibodies. Polyclonal antibodies canbe obtained by recovering antibodies to the target antigen from animmunized animal. Alternatively, monoclonal antibody-producing cells canbe obtained by cloning an antibody-producing cell derived from animmunized animal.

Furthermore, by using antibodies or genes thereof obtained from animmunized animal such as mice, chimeric antibodies of human andimmunized animals, or humanized antibodies can be obtained. Methods forproducing these antibodies that comprise modified structures are alsoknown in the art.

Furthermore, this invention relates to the antibodies obtained by themethods of the present invention. The antibodies of the presentinvention comprise any kind of antibody that can be obtained by aprocedure comprising a method as described above. Consequently, thisinvention includes, for example, monoclonal antibodies, polyclonalantibodies, chimeric antibodies of human and immunized animals,humanized antibodies, and human antibodies. For example, a transgenicmouse whose immune system has been substituted with that of a human isknown in the art. Human antibodies can be obtained by immunizing suchmice.

Preferable antibodies in the present invention are antibodies thatrecognize human membrane proteins. Many membrane proteins are importantas target molecules for drug discovery. However, antibodies specific tomembrane proteins have been considered difficult to obtain due topurification difficulties. The present invention, however, has made itpossible to efficiently obtain a subject antibody, even though thetarget antigen is a recombinantly-produced membrane protein thatco-exists with a background antigen. For example, as a membrane protein,PepT1 is an important molecule. The nucleotide sequence and amino acidsequence of PepT1 are already known: human PepT1 (GenBank XM_(—)007063)is described in J. Biol. Chem. 270(12): 6456-6463 (1995); and mousePepT1 (GenBank AF205540) is described in Biochim. Biophys. Acta. 1492:145-154 (2000)).

Those anti-PepT1 antibodies that bind to an extracellular region ofPepT1 are useful. In particular, an antibody that specifically binds toan extracellular region of PepT1 is preferable in the present invention.In the present invention, the phrase “specifically binds to anextracellular region” means the ability to immunologically discriminateextracellular regions of PepT1 from other regions. More specifically, anantibody that specifically binds to an extracellular region of PepT1 isdefined as an antibody that binds to an extracellular region, but doesnot bind, for example, to an intracellular region or transmembranedomain of PepT1. Human PepT1 is a preferable PepT1 in the presentinvention. Human PepT1 includes not only PepT1s derived from humans, butalso recombinant PepT1s obtained by expressing human PepT1 in abaculovirus expression system.

The human PepT1 molecules that are used as immunogens do not have to beentire molecules, as long as they retain a target antigen structure. Forexample, a fragment comprising a PepT1 extracellular region can be usedas an immunogen. A preferable PepT1 in the present invention is a humanPepT1 that comprises transport activity, or a full-length human PepT1. Afull-length human PepT1 comprising transport activity is especiallypreferable. The transport activity of a human PepT1 can be detected byusing the activity of incorporating a substrate into a cell as anindicator. As its substrates, PepT1 is known to incorporateglycylsarcosine or such into cells. Incorporation of glycylsarcosine canbe assayed by using [¹⁴C] glycylsarcosine or such.

Human PepT1 is preferably expressed on the surface of a membrane (suchas a viral envelope or cell membrane). The transport activity of a PepT1expressed on the surface of a viral envelope can be detected bycontacting a solution comprising viral particles with a substrate; andthen monitoring the incorporation of the substrate into the viralparticles.

Well-known methods can be used for the methods of immunizing to obtainantibodies. Animals can be immunized with an immunogen using knownmethods. General methods include injecting a sensitizing antigen into amammal by subcutaneous or intraperitoneal injection. Specifically, animmunogen is diluted with an appropriate volume of Phosphate-BufferedSaline (PBS) or physiological saline, and as desired, the suspension ismixed with an appropriate volume of a conventional adjuvant. This isemulsified and applied to the mammals. For example, Freund's completeadjuvant can be used as an adjuvant. In addition, after this, animmunogen that has been mixed with an appropriate volume of Freund'sincomplete adjuvant is preferably applied several times every four to 21days.

When immunizing an immunogen, an appropriate carrier can also be used.In this way immunization occurs, and the increased level of a desiredantibody in the serum can be confirmed using conventional methods.

When obtaining the target antibodies, an increase in the level of adesired antibody in the serum is confirmed, and blood is then collectedfrom the immunized mammals. Serum can be separated from collected bloodusing known methods. As polyclonal antibodies, serum comprisingpolyclonal antibodies can be used. Where necessary, fractions comprisingpolyclonal antibodies can be isolated from this serum, and this fractioncan also be used.

For example, fractions only recognizing the target antigens can beobtained using affinity columns coupled to the target antigens.Immunoglobulin G or M can be prepared by purifying these fractions usinga protein A or protein G column.

After confirming the increase in the level of the intended antibody inthe serum of a mammal that was sensitized by the above-describedantigen, the antibody-producing cells are extracted from the mammal andcloned to obtain monoclonal antibodies. Spleen cells and such can beused as antibody-producing cells. Antibody-producing cells can be clonedby cell fusion methods. Mammalian myeloma cells and such can be used asparent cells to be fused with the above-mentioned antibody-producingcells. Even more preferably, myeloma cells that comprise uniqueauxotrophy or drug resistance can be examples of useful selectivemarkers for fusion cells (hybridoma cells).

By basically following the methods known in the art, fusion cells can beobtained from the antibody-producing cells and the myeloma cellsdescribed above. Methods for producing monoclonal antibodies by usingthe cell fusion techniques have been established, for example, byMilstein et al. (Galfre, G. and Milstein, C., Methods Enzymol. (1981)73, 3-46).

The hybridoma cells produced by cell fusion techniques are selected byculturing in a selective medium. A suitable selective medium can be usedin accordance with the characteristic features of the myeloma cells usedfor the cell fusion. HAT medium (a medium comprising hypoxanthine,aminopterine, and thymidine), for example, can be used as a selectivemedium. The hybridoma cells are cultured in the HAT medium for a timesufficient to kill all cells other than the intended hybridoma cells(e.g. all non-fused cells). Generally, hybridoma cells can be selectedby continuing culture for several days to several weeks. Afterselection, a standard limiting dilution method can be used to screen andclone the hybridoma cells that produce the subject antibodies.

Subsequently, the hybridoma cells thus obtained are intraperitoneallytransplanted into mice to obtain ascites fluid comprising the monoclonalantibodies. Monoclonal antibodies can also be purified from the ascitesfluid. For example, monoclonal antibodies can be purified by ammoniumsulfate precipitation methods, protein A or protein G columns, DEAE ionexchange chromatography, or affinity columns coupled with a targetantigen.

In addition to producing antibodies by using hybridomas,antibody-producing cells such as antibody-producing sensitizedlymphocytes and such, which have been immortalized using oncogenes orviruses and such, can also be used. Epstein-Barr virus (EBV) and so oncan be used as a virus for immortalizing cells.

Monoclonal antibodies obtained in this way can also be used asrecombinant antibodies that were produced using gene recombinationtechnologies (for example, see Borrebaeck, C. A. K. and Larrick, J. W.,Therapeutic Monoclonal Antibodies, UK, Macmillan Publishers Ltd., 1990).Recombinant antibodies can be produced by cloning the DNAs that encodethem from antibody-producing cells, such as hybridomas andantibody-producing sensitized lymphocytes, then incorporating these DNAsinto a suitable vector, and introducing this vector into a host. Thepresent invention also encompasses such recombinant antibodies.

The antibodies obtained by the methods of the present invention can alsobe antibody fragments, modified antibodies, and the like. For example,an antibody fragment can be an Fab, F(ab′)₂, Fv, or a single chain Fv(scFv) where the Fvs of an H chain and L chain are linked by a suitablelinker (Huston, J. S. el al., Proc. Natl. Acad. Sci. U.S.A., (1998) 85,5879-5883). Specifically, the antibody fragments can be obtained bytreating antibodies with an enzyme such as papain or pepsin.Alternatively, genes encoding these antibody fragments are constructed,inserted into an expression vector, and expressed in appropriate hostcells (see for example, Co, M. S. at al., J. Immunol. (1994) 152,2968-2976; Better, M. and Horwitz, A. H., Methods Enzymol. (1989) 178,476-496; Pluckthun, A. and Skerra, A., Methods Enzymol. (1989) 178,497-515; Lamoyi, E., Methods Enzymol. (1986) 121, 652-663; Rousseaux, J.et al., Methods Enzymol. (1986) 121, 663-669; Bird, R. E. and Walker, B.W., Trends Biotechnol. (1991) 9, 132-137).

Antibodies bound to various molecules such as polyethylene glycols(PEG), can also be used as the modified antibodies. “Antibody” in thepresent invention also encompasses these modified antibodies. Suchmodified antibodies can be obtained by chemically modifying obtainedantibodies. These methods have already been established in the art.

In addition, methods for obtaining human antibodies are known. A targetantibody can be obtained by immunizing transgenic animals, that comprisethe entire repertoire of human antibody genes, with a target antigen(see, International Patent Application No. WO 93/12227, WO 92/03918, WO94/02602, WO 94/25585, WO 96/34096, and WO 96/33735).

The antibodies obtained by the methods of the present invention can bechimeric antibodies comprising non-human antibody-derived variableregions, derived from the immunized animals, and human antibody-derivedconstant regions. In addition, they can also be humanized antibodiescomprising non-human antibody-derived complementarity determiningregions (CDRs) which are derived from the immunized animals, humanantibody-derived framework regions (FRs), and constant regions.

These modified antibodies can be produced using known methods.Specifically, for example, a chimeric antibody is an antibody comprisingthe antibody heavy chain and light chain variable regions of animmunized animal, and the antibody heavy chain and light chain constantregions of a human. A chimeric antibody can be obtained by (1) ligatinga DNA encoding a variable region of an immunized animal-derived antibodyto a DNA encoding a constant region of a human antibody; (2)incorporating this into an expression vector; and (3) introducing thevector into a host for production of the antibody.

A humanized antibody, which is also called a reshaped human antibody, isa modified antibody. A humanized antibody is constructed bytransplanting a complementarity determining region (CDR) of an antibodyof an immunized animal, into the CDR of a human antibody. Conventionalgenetic recombination techniques for the preparation of such antibodiesare known.

Specifically, a DNA sequence designed to ligate a mouse antibody CDRwith a human antibody framework region (FR) is synthesized by PCR, usingseveral oligonucleotides constructed to comprise overlapping portions attheir ends. A humanized antibody can be obtained by (1) ligating theresulting DNA to a DNA which encodes a human antibody constant region;(2) incorporating this into an expression vector; and (3) transfectingthe vector into a host to produce the antibody (see, European PatentApplication No. EP 239,400, and International Patent Application No. WO96/02576). Those human antibody FRs that are ligated via the CDR, suchthat the CDR forms a favorable antigen-binding site, are selected. Asnecessary, amino acids in the framework region of an antibody variableregion may be substituted such that the CDR of a reshaped human antibodyforms an appropriate antigen-binding site (Sato, K. et al., Cancer Res.(1993) 53, 851-856).

Furthermore, genes coding for the antibodies can be isolated from theantibody-producing cells of an immunized animal. Methods used to isolategenes that code for antibodies are not limited. For example, genescoding for antibodies can be obtained by amplification using the PCRmethod, by using as templates those genes that code for variableregions, CDRs, or the like. Primers for the amplification of genes thatcode for antibodies are known in the art. Subject antibodies can beproduced by expressing genes thus obtained in an appropriate expressionsystem. Alternatively, the genes obtained by the present invention canbe used to produce various modified antibodies, as described above.

Antibodies obtained as above can be purified until they are homogenousimmunoglobulin molecules. These purification methods are notparticularly limited. Separation and purification methods conventionallyused for polypeptides can be used to separate and purify the antibodiesused in the present invention. For example, immunoglobulins can beseparated and purified by appropriately selecting and combiningchromatography columns such as affinity chromatography columns, filters,ultrafiltration, salt precipitation, dialysis, SDS polyacrylamide gelelectrophoresis, isoelectric focusing and so on (Antibodies: ALaboratory Manual. Ed Harlow and David Lane, Cold Spring HarborLaboratory, 1988). The concentration of the above-obtained antibodiescan be determined by measuring absorbance, or by enzyme-linkedimmunosorbent assays (ELISA), etc.

Protein A columns, protein G columns, and such can be used as thecolumns for use in affinity chromatography. For example, Hyper D, POROS,Sepharose F.F. (Pharmacia) and so on are examples of the columns usingprotein A.

Examples of chromatography other than affinity chromatography includeion exchange chromatography, hydrophobic chromatography, gel filtration,reverse chromatography, and adsorption chromatography (Strategies forProtein Purification and Characterisation: A Laboratory Course Manual.Ed Daniel R, Marshak et al., Cold Spring Harbor Laboratory Press, 1996).These chromatographies can be carried out using liquid phasechromatography such as HPLC and FPLC.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 and 2 show the nucleotide sequence of the constructed gp64 gene.

FIG. 3 shows the structure of the pCAG-gp64 vector constructed inExample 1.

FIG. 4 is a photograph of the Founder mice testes.

FIG. 5 is a photograph showing the result of mRNA expression analysis byNorthern blotting. In this figure, H, B, I, and M refer to heart, brain,intestine, and muscle, respectively.

FIG. 6 is a photograph showing the results of Western blotting analysisusing anti-mouse IgG. In this figure, “pre” and “2nd” respectively referto pre-immunization blood collection, and blood collection after thesecond immunization. Gp64TgM and wtBALB/c represent transgenic andnon-transgenic mice, respectively.

FIG. 7 is a photograph showing the results of Western blotting analysisusing anti-mouse IgG. Gp64TgM and wtBALB/c represent transgenic andnon-transgenic mice, respectively.

FIG. 8 shows the result of FACS analysis of the antibody titer forPepT1-specific antibody in the mouse serum. In this figure, the x-axisand y-axis respectively represent cell number (log scale) andfluorescence intensity. (Above) mouse #1; (below) mouse #2.

FIG. 9 shows the results of the same analysis in FIG. 8. (Above) mouse#3; (below) no antibody.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention is specifically described herein below usingExamples, however, it is not to be construed as being limited thereto.

Example 1 Construction of gp64 Transgenic Vector

The nucleotide sequence of gp64 and the amino acid sequence encoded bythe gp64 gene are shown in SEQ ID NOs: 3 and 4, respectively (GenBankAcc No. 9627742). PCR was carried out using the gp64 gene as a template,and using the following primer set: the 5′ primer 64F1 (SEQ ID NO: 1),which comprises an EcoRI recognition sequence and the KOZAK sequence atits 5′ terminus; and the 3′ primer 64R1 (SEQ ID NO: 2), which comprisesan EcoRI recognition sequence at its 5′ terminus (FIGS. 1 and 2). ThePCR conditions are shown below:

The PCR reaction solution composition was 5 μl of ×10 ExTaq buffer, 4 μlof dNTP supplied with ExTaq, 1 μl of 10 μmol/l 64F1 primer, 1 μl of 10μmol/l 64R1 primer, 1 μl of 500 pg/μl pBac-N-blue, 0.5 μl of 5 units/μlExTaq, and 37.5 μl of deionized water (DIW). PCR was carried out for:

5 minutes at 94° C.;25 cycles of “15 seconds at 94° C., 30 seconds at 57° C., and 30 secondsat 72° C.”;7 minutes at 72° C.; and4° C. forever.

The amplified band was subcloned into pGEM-Teasy, and then transformedE. coli. DH5α cells. After performing colony PCR using T7 and SP6primers, the nucleotide sequence of clones confirmed to comprise theinsert was analyzed with the ABI Prism® 377 DNA sequencer (AppliedBiosystems) and the BigDye® Terminator Cycle Sequencing kit (AppliedBiosystems), in combination with the T7 primer or the SP6 primer. As aresult, clones comprising the subject gene were confirmed. A fragmentcomprising the gp64 gene and confirmed to comprise no mutations in itsnucleotide sequence was isolated from the clones by EcoRI digestion, andthen inserted into an EcoRI-digested pCAGGS1. The resulting vector wasused to transform E. coli DH5α cells. Cells comprising the clone asdesigned were incubated in 250 ml of LB medium at 37° C. overnight, andpurified by using the Endofree MAXI kit (QIAGEN) to obtain 581.6 μg ofplasmid.

Example 2 Introduction of the Gene

The DNA fragment for injection was prepared as follows: The pCAGGSvector into which the gp64 gene was inserted (pCAG-gp64, FIG. 3) wastreated with SalI and PstI to yield a fragment (about 3.8 kb) comprisingthe gp64 gene. This fragment (about 3.8 kb) was extracted using the GelExtraction kit (QIAGEN), and then diluted with PBS to a concentration of3 ng/μl, yielding the DNA fragment for injection.

The mouse pronuclear eggs to be injected with the DNA fragment werecollected as follows: Specifically, BALE/c series female mice (NipponCLEA) were induced to superovulate by intraperitoneal administration of5 international units (i.u) of pregnant mare serum gonadotrophin (PMSG),followed by intraperitoneal administration of 5 i.u of human chorionicgonadotrophin (hCG) 48 hours later. These female mice were mated withmale mice of the same lineage. The morning after mating, the oviducts offemale mice that were confirmed to have a vaginal plug were perfused torecover pronuclear eggs.

The DNA fragments were injected into the pronuclear eggs with amicromanipulator (Experimental Medicine (Jikken Igaku) suppl., thelatest technologies in gene targeting (gene targeting no saishingijyutu) (Yodosha), 190-207, 2000. The DNA fragments were injected into373 embryos of BALB/c mice. On the next day, 216 embryos that haddeveloped to the two-cell stage were transplanted into the oviducts ofrecipient female mice, which were in the first day of pseudopregnancy,at a density of around ten embryos per oviduct (i.e. around 20 embryosper mouse).

The recipient female mice that did not give birth to offspring by theexpected date of delivery were subjected to caesareans, and theresultant offspring were brought up by a foster parent. The results aresummarized in Table 1. Fifty offspring were obtained, four of which weretransgenic mice into which the gp64 gene has been introduced (referredto as Tgm below). Hereinafter, the transgenic mice obtained in the firstgeneration are described as “Founder” mice.

TABLE 1 Viable embryos after injection/ Transplanted Implanted OffspringWeaned Offspring Embryos receiving injection embryos embryos (female,male) (female, male) Founder 1st 59/63 55 20  9 (4, 5)  9 (4, 5) 0 2nd186/223 161 57 26 (13, 13) 25 (13, 12) Male 3 3rd 61/87 56 35 15 (9, 6)15 (9, 6) Male 1 Total 306/373 216 107 50 (25, 25) 49 (25, 24) Male 4

All of the four Founder mice were male. Two lines (Nos. 30 and 31) ofthese four resulted in four and 20 offspring (F1 mice), respectively.The F1 mice thus obtained were genotyped, and three offspring in line 30were found to be Tgm, indicating inheritance of the gp64 gene to thesecond generation. On the other hand, in line 31, all 20 offspring werefound to be wild type mice (non-Tgm), in which the gp64 gene could notbe detected. Accordingly, the gp64 gene was considered to be integratedinto the line 31 Founder mouse in a mosaic structure. Founder mice oflines 34 and 46 had no fertility properties, and therefore, offspringwere not obtained. Although the Founder mouse of line 30 impregnated onerecipient female immediately after the initiation of crossing, nofurther offspring was obtained after that (Table 2).

TABLE 2 Offspring obtained Line Date of Copy Number of the (date ofbirth, No. birth Sex introduced gene total offspring, and Tgm) Notes 30010709 Male More than 10 010926 Female 3, Female 3 No offspring werecopies Male 1 obtained after the first delivery. Testes are small andsperm are not observed. 31 010709 Male 2 to 3 copies 010927 Female 3, 0Mosaic for gene Male 5 transfer 011022 Male 2 0 011108 Female 4, 0 Male6 34 010709 Male 2 to 3 copies No fertility — — Testes are small andproperties sperm are not observed. 46 010821 Male 2 to 3 copies Nofertility — — Testes are small and properties sperm are not observed.

Consequently, sperm from the Founder mice of lines 30, 34, and 46 wasextracted in order to carry out in vitro fertilization. The testes ofall three Founder mice were abnormally small (FIG. 4), and no sperm wasobserved in their cauda epididymidis. Thus in vitro fertilization couldnot be achieved. From these results, the gp64 protein was found toaffect the spermatogenic ability of mice. Therefore, it may be possibleto use gp64 in contraception and such.

Example 3 Confirmation of the Introduced Gene

DNA was extracted from tails of three week-old mice using an automatednucleic acid isolation system (KURABO), and the presence of theintroduced gene was confirmed by Southern blotting method and PCR. Theintroduced gene was confirmed by Southern blotting, as follows: First,15 μg of genome DNA was digested with EcoRI, subjected toelectrophoresis, and transferred to a nylon membrane. Then, the presenceof the introduced gene was confirmed by hybridizing the transferred DNAwith a probe, which was about 1.5 kb of EcoRI-digested fragment ofpCAG-gp64 vector that comprises the gp64 gene. The presence of theintroduced gene was also confirmed by the PCR method, using about 100 ngof DNA as a template, and primers comprising the sequences as shownbelow:

(SEQ ID NO: 1) Sense primer 64F1: GAATTCCACCATGGTAAGCGCTATTGTT; and (SEQID NO: 2) Antisense primer 64R1: GAATTCTTAATATTGTCTATTACGGT.PCR was carried out for:5 minutes at 94° C.;35 cycles of “15 seconds at 94° C., 30 seconds at 57° C., and 30 secondsat 72° C.”;7 minutes at 72° C.; and4° C. forever.

The PCR products thus obtained were subjected to electrophoresis toconfirm the introduced gene using the presence or absence of a bandcorresponding to about 1.5 kb as an indicator.

Example 4 Confirmation of the Expression of the gp64 Gene in gp64 Tgm

In the line 30 Founder mouse in which inheritance of the gp64 gene tothe second generation had been confirmed, expression of the gp64 genewas confirmed by Northern blotting analysis. Specifically, total RNA wasextracted from four kinds of organ, heart, brain, intestine, and a thighmuscle, by using ISOGEN (Nippon Gene). Then, 20 μg of the total RNA wassubjected to electrophoresis, and was transferred to a nylon membrane.An about 1.5 kb of EcoRI-digested fragment of pCAG-gp64 vector thatcomprises the gp64 gene was used as the probe for the Northern blottinganalysis. An around 1.5 kb band corresponding to the gp64 gene wasexpected from the vector construct.

FIG. 5 shows these results. Expression of the gp64 gene was confirmed atleast in heart, brain, and thigh muscle. The reason why the bands wereseen as three bands is unknown.

Example 5 Fertility Properties of the Line 30 Female Tgm (Crossing ofMice)

When the line 30 female Tgm turned eight weeks-old, they were crossedwith a male mouse of the same lineage.

As a result, a total of 31 (14 females and 17 males) offspring (F2) wereobtained from two deliveries by each of the three F1 female mice (Table3). 14 of these offspring (five females and nine males) were Tgm. Sinceoffspring were also obtained from the third delivery, the female Tgmwere shown to have normal fertility properties.

TABLE 3 Individual Number of Offspring Offspring Sex Number Deliveries(Non-Tgm) (Tgm) Female 1 2 Female 3, Female 1, Male 1 Male 6 Female 2 2Female 4, Female 2, Male 3 Male 1 Female 3 2 Female 2, Female 2, Male 4Male 2

Example 6 Preparation of Budding Baculoviruses Expressing PepT1

Budding baculoviruses expressing PepT1 and used as immunogens wereprepared as follows: PepT1 is a membrane protein that acts as atransporter. The PepT1 structure is known in the art (GenBankXM_(—)007063, J. Biol. Chem. 270(12): 6456-6463 (1995)).

A full-length PepT1 gene was isolated from a human kidney library usingPCR. By inserting the full-length human PepT1 gene into pBlueBacHis2A(Invitrogen), the pBlueBacHis-PepT1 transfer vector was constructed. ABac-N-Blue transfection kit (Invitrogen) was then used to introduce thistransfer vector into Sf9 cells, along with Bac-N-Blue DNA. Thus, arecombinant virus for the expression of human PepT1 was constructed.Specifically, 4 μg of pBlueBacHis-PepT1 was added to Bac-N-Blue DNA, andthen 1 mL of Grace's medium (GIBCO) and 20 μL of cell FECTIN reagent wasadded. This was mixed, incubated for 15 minutes at room temperature, andthen added drop-by-drop to 2×10⁶ Sf9 cells washed once with Grace'smedium. After incubating for four hours at room temperature, 2 mL ofcomplete medium (Grace's medium which comprises 10% fetal bovine serum(Sigma), 100 units/mL penicillin, and 100 μg/mL streptomycin(GIBCO-BRL)) was added and cultured at 27° C. Recombinant viruses forexpressing human PepT1, which were constructed by homologousrecombination, were cloned twice according to the instructions attachedto the kit. A virus stock of the recombinant viruses was thus obtained.

Construction of budding-type viruses that express human PepT1 wascarried out as follows: Specifically, 500 mL of Sf9 cells (2×10⁶/mL)were infected with the recombinant viruses prepared as above, so as toachieve MOI=5. After culturing at 27° C. for three days, the culturesupernatant was centrifuged for 15 minutes at 800×g, and the cells andcell debris were removed. The supernatant recovered by centrifugationwas centrifuged at 45,000×g for 30 minutes, and the precipitate was thensuspended in PBS. The cellular components were removed by centrifugingfor another 15 minutes at 800×g. The supernatant was again centrifugedat 45,000×g for 30 minutes, and the precipitate was again suspended inPBS. This suspension was the budding virus fraction. Expression of PepT1in the virus and on the Sf-9 cell membrane was confirmed by Westernanalysis using anti-His antibodies. In addition, protein concentrationwas measured using Dc Protein Assay kit (Bio-Rad), with BSA as thestandard.

Example 7 Immunization of Mice

Mice were immunized by subcutaneous injection with an immunogen, whichwas emulsified according to the standard method using complete andincomplete Freund's adjuvants (Difco). Injection doses in the first andthe second immunizations were 1 mg/mouse and 0.5 mg/mouse, respectively.The second immunization was given 14 days after the first immunization.Seventeen days after the first immunization, serum samples were takenfrom the mice by retro-orbital bleeding.

Example 8 Confirmation of Immunotolerance to gp64 by Western BlottingAnalysis

PepI1 expressing budded baculovirus (PepT1-BV) (1 μg/lane) was subjectedto SDS-PAGE analysis on 12% gel under reducing conditions. After theelectrophoresis, proteins were electroblotted onto a polyvinylidenedifluoride (PVDF) membrane. This membrane was reacted with 1,000fold-diluted serum samples, sequentially washed, and then reacted with a1,000 fold-diluted Biotin-Anti-Mouse IgG(γ) (Zymed) andStreptavidin-Alkaline Phosphatase (Zymed). An alkaline phosphatasestaining kit (Nakarai Tesque) was used for staining. A positive controlantibody for detecting gp64 was purchased from NOVAGEN.

FIG. 6 shows the results. When stained with the Anti-Mouse IgG, a bandcorresponding to the gp64 protein was strongly stained for the lanesreacted with both of the two serum samples obtained from non-transgenicmice. On the other hand, though gp64 was detected in all three gp64transgenic mice, staining was weak. These results indicate that theamount of anti-gp64 antibody produced by the transgenic mice isconsiderably less than that produced by non-transgenic mice. Althoughthe Anti-Mouse IgM staining was weak for the two non-transgenic mice, itwas very weak or not stained at all in the gp64 transgenic mice (FIG.7).

Example 9 Production of Anti-PepT1 Antibodies by gp64 Tgm

Following procedures were used for the initial immunization. 200 μl PBScomprising 1 mg of PepT1-BV and 100 ng of pertussis toxin wassubcutaneously injected into Tgm. For the second and subsequentimmunizations, 0.5 mg of PepT1-BV suspension in PBS was subcutaneouslyinjected.

Ba/F3 cells expressing PepT1 on the cell surface (herein after, referredto as Ba/F3-PepT1) and Ba/F3 cells expressing no PepT1 were washed twicewith PBS, respectively. 100 μl of mouse serum sample that was 220fold-diluted with PBS was added to 1×10⁶ cells of each cell type,followed by reaction for 30 minutes on ice. After reaction, cells werewashed once with 500 μl PBS and 100 μl of FITC-anti-mouse IgG 200fold-diluted with PBS was added. This was allowed to react for 30minutes on ice. After centrifugation, cells were suspended in 500 μl ofPBS and analyzed by FACS. FIGS. 8 and 9 show the results of FACSanalysis of a serum obtained from a mouse after the fifth immunization.In these figures, solid lines and dotted lines indicate Ba/F3 andBa/F3-PepT1 cells, respectively.

From these results, the titer of antibody reacting specifically withPep-T1 was confirmed to be increased in the serum of mice immunized withPepT1-BV.

INDUSTRIAL APPLICABILITY

This invention enables efficient production of antibodies against targetantigens, using target antigens that comprise background antigens. Theantibody-production methods of the present invention are useful inproducing antibodies by using immunogens in which contamination bybackground antigens is inevitable.

For example, an exogenous gene expression system, known as thebaculovirus expression system, is useful as a tool for obtainingrecombinant proteins easily and in large quantities. In particular, whenapplied to membrane proteins, the baculovirus expression system isexcellent in that the membrane proteins are obtainable with other viralenvelope proteins in a state that maintains their structure. However,this expression system is also problematic in that, when using thisexpression product as the immunogen, gp64 acts as a background antigenand interferes with the acquisition of antibodies against a targetantigen.

By using the antibody-production methods of the present invention, it ispossible to efficiently suppress the adverse effect of backgroundantigens on the proteins prepared by the baculovirus expression system.As a result, anti-membrane protein antibodies can be producedefficiently by using the membrane protein antigens, which can beobtained in large quantities using the baculovirus expression system, astarget antigens.

Membrane proteins include many functionally important proteins such asreceptors and cell adhesive proteins. Therefore, antibodies thatrecognize membrane proteins are expected to play an important role infunctional analysis, localization analysis, quantification, diagnosis,or the development of therapeutic agents that regulate membrane proteinactivities.

Preparation of Membrane Proteins Applicable as Immunogens has beenthought to be difficult. However, by the present invention, largequantities of membrane proteins produced by, for example, thebaculovirus expression system, can be used as immunogens withoutremoving background antigens. Consequently, many antibodies thatrecognize various membrane proteins and that have been considered to bedifficult to produce can now obtained very efficiently.

The antibody-production methods of the present invention contribute tothe functional analysis of membrane proteins and diagnosis usingantibodies, and to the development of drugs based on the regulation ofmembrane protein activities.

All prior art documents cited in the present application are herebyincorporated by reference in their entirety.

1. A method for producing an antibody that recognizes a target antigen,wherein the method comprises the steps of: i) immunizing a non-humananimal that has immunotolerance to a background antigen comprised in animmunogen, wherein the immunogen comprises both the target antigen andthe background antigen; and ii) obtaining an antibody against the targetantigen, or a gene encoding the antibody.
 2. The method of claim 1,wherein immunotolerance is induced artificially.
 3. The method of claim1, wherein the non-human animal is a transgenic non-human animal.
 4. Amethod for producing an antibody against a target antigen, wherein themethod comprises the steps of: (a) preparing an immunogen comprising thetarget antigen and a background antigen; (b) producing a transgenicnon-human animal comprising a gene expressibly encoding the backgroundantigen; (c) administering the immunogen of (a) to the transgenicnon-human animal of (b); and (d) isolating the antibody against thetarget antigen from the transgenic non-human animal.
 5. The method ofclaim 4, wherein the immunogen is a virus particle or a part thereof. 6.The method of claim 5, wherein the virus is a baculovirus.
 7. The methodof claim 4, wherein the target antigen is a membrane protein.
 8. Themethod of claim 6, wherein the background antigen is gp64.
 9. The methodof claim 4, wherein the non-human animal is a mouse.
 10. An antibodythat is produced by the method of any one of claims 1 to
 9. 11. Achimeric antibody between a non-human animal and human, or a humanizedantibody, produced using the antibody of claim
 10. 12. A transgenicnon-human animal, into which a gene encoding a viral envelope protein isintroduced.
 13. The transgenic non-human animal of claim 12, wherein thevirus is a baculovirus.
 14. The non-human animal of claim 13, whereinthe viral envelope protein is gp64.
 15. The non-human animal of claim12, wherein the non-human animal is a mouse.
 16. The non-human animal ofclaim 12, for use in producing an antibody against an antigen comprisinga viral protein.
 17. A method for producing a non-human immunizedanimal, wherein the method comprises the step of producing a transgenicnon-human animal into which a gene encoding a background antigen isintroduced.
 18. A non-human immunized animal for obtaining an antibodyagainst a target antigen comprising a background antigen, wherein theanimal is produced by the method of claim
 17. 19. A method for producingan antibody against PepT1, wherein the method comprises the steps of:(a) preparing a baculovirus that expressibly comprises a DNA whichencodes PepT1 or a fragment thereof; (b) infecting a host cell with thebaculovirus of (a) to obtain a budding virus that expresses PepT1 or afragment thereof; (c) producing a transgenic non-human animal thatexpressibly comprises a gene encoding a baculovirus membrane proteingp64; (d) immunizing the transgenic non-human animal of (c) with afraction comprising the budding virus of (b) or PepT1 or its fragment;and (e) recovering the antibody-recognizing PepT1 from the immunizedanimal.